1. Field of the Invention
The present invention relates generally to the field of plant biology. More particularly, the present invention is directed to compositions and methods for use in regulation of plant growth.
This invention was made with Government support under Grant No. DCB-90-16756 and INB-94-06974, awarded by the National Science Foundation, and Grant No. 90-00070, awarded by the United States Department of Agriculture. The Government has certain rights in this invention.
2. Description of the Related Art
Present strategies for controlling plant developmental and physiological functions rely on traditional genetic approaches, or on biotechnological approaches that lack a fully refined conceptual foundation. In terms of manipulation of plant resource allocation, the only approach currently available involves the use of genetic breeding for a desired trait; this is recognized as a slow and complex process. Furthermore, current strategies fail to provide any understanding of the underlying molecular events that are involved in the prioritization of resource allocation to the various regions of the plant body.
Partitioning of assimilates in plants is an important and highly regulated process [Wardlaw I. F. (1990) The control of carbon partitioning in plants. New Phytologist 116, 341-381]. It involves regulation of photosynthesis, intracellular and intercellular transport of metabolites, phloem loading and unloading, storage and other interrelated biochemical processes. Partition of assimilates is closely related to the regulation of growth and development, in as much as growth of different plant parts and organs often requires the import of assimilates from elsewhere in the plant. The relationship between root and shoot biomass is an excellent example of regulation of partition of assimilates. Root-to-shoot ratios vary from plant species-to-species, and are influenced by the environment [Geiger D. R. and Servaites J. S. (1991) Carbon allocation and response to stress. In Response of plants to multiple stresses (eds. H. A. Mooney, W. E. Winner and E. J. Pell ) pp. 103-127. Academic Press, New York; Mooney H. A. and Winner W. E. (1991) Partitioning response of plants to stress. In Response of plants to multiple stresses (eds. H. A. Mooney, W. E. Winner and E. J. Pell) pp. 129-141. Academic Press, New York]. Furthermore, this ratio is responsive to water stress and nutrient deficiencies, and it can be manipulated by exogenous hormonal treatments and light quality [Britz S. J. (1990) Photoregulation of root: shoot ratio in soybean seedlings. Photochemistry and Photobiology 52, 151-159; Incoll L. D., Ray J. P. and Jewer P. C. (1990) Do cytokinins act as root to shoot signals? In Importance of root to shoot communication in the responses to environmental stress, Monograph 21 (eds. W. J. Davies and B. Jeffcoat) pp. 185-199. British Society for Plant Growth Regulation, Bristol; Davies W. J. and Zhang J. (1991) Root signals and the regulation of growth and development of plants in drying soil. Annual Review of Plant Physiology and Plant Molecular Biology 42, 55-76; Tolley-Henry L. and Raper C. D. (1991) Soluble carbohydrate allocation to roots, photosynthetic rate of leaves and nitrate assimilation as affected by nitrogen stress and irradiance. Botanical Gazette 152, 23-33].
The finding [Lucas W. J., Olesinski A., Hull R. J., Haudenshield J. S., Deom C. M., Beachy R. N. and Wolf S. (1993) Influence of the tobacco mosaic virus 30-kDa movement protein on carbon metabolism and photosynthate partitioning in transgenic tobacco plants. Planta 190, 88-96] that a significant reduction in biomass partitioning to roots occurs in transgenic tobacco plants that express the tobacco mosaic virus movement protein (TMV-MP) has raised questions as to the possible effects of this protein on the integrated physiology of tobacco plants. It is now well established that the TMV-MP interacts with plasmodesmata to potentiate virus trafficking between cells [Deom C. M., Oliver M. J. and Beachy R. N. (1987) The 30-kDa gene product of tobacco mosaic virus potentiates virus movement. Science 237, 389-394; Wolf S., Deom C. M., Beachy R. N. and Lucas W. J. (1989) Movement protein of tobacco mosaic virus modifies plasmodesmatal size exclusion limit. Science 246, 377-379; Ding B., Haudenshield J. S., Hull R. J., Wolf S., Beachy. R. N. and Lucas W. J. (1992) Secondary plasmodesmata are specific sites of localization of the tobacco mosaic virus movement protein in transgenic tobacco plants. Plant Cell 4, 915-928; Waigmann E., Lucas W. J., Citovsky V. and Zambryski P. (1994) Direct functional assay for tobacco mosaic virus cell-to-cell movement protein and identification of a domain involved in increasing plasmodesmal permeability. Proc. Natl. Acad. Sci. USA 91, 1433-1437]. In transgenic tobacco plants expressing the TMV-MP, the size exclusion limit (SEL) of plasmodesmata connecting the mesophyll and bundle sheath cells was found to be increased from 800 Da to greater than 9.4 kDa [Wolf et al. 1989, supra; Deom C. M., Wolf S., Holt C. A., Lucas W. J. and Beachy R. N. (1991) Altered function of the tobacco mosaic virus movement protein in a hypersensitive host. Virology 180, 251-256; Ding et al. 1992, supra]. This observation raised the possibility that dilated plasmodesmata, within such tissues, may enhance symplasmic carbohydrate transport between cells [Lucas W. J. and Wolf S. (1990) Plasmodesmatal function probed using transgenic tobacco plants. In Recent advances in Phloem transport and assimilate compartmentation (eds. J. L. Bonnemain, J. Dainty, S. Delrot and W. J. Lucas) pp. 106-115. Ouest Editions, Nantes Cedex; Lucas et al. 1993, supra]. However, contrary to this expectation, these transgenic plants exhibited a decrease in translocation of assimilates, from source leaves, during the day [Lucas et al. 1993, supra].
Also, in such transgenic plants expressing the TMV-MP, root-to-shoot ratios were significantly smaller, reflecting reduced carbon allocation and translocation to the roots [Lucas et al. 1993, supra]. It is thus of great interest that the TMV-MP affects not only the dilation of plasmodesmata and virus trafficking, but also carbohydrate metabolism and resource allocation, as reflected by changes in root-to-shoot ratios.
Similar considerations are involved in understanding the distribution of other plant products, such as sucrose. Sucrose synthesis occurs within the cytosol of tobacco mesophyll cells, but the pathway followed by sucrose during its movement from the site of synthesis to the cells of the phloem remains equivocal. The prevailing view is that solute movement between mesophyll cells occurs via a symplasmic route through plasmodesmata [Giaquinta, R. T. (1983) Phloem loading of sucrose. Ann. Rev. Plant PhysioL 34, 347-387; Tucker, J. E., Mauzerall, D., Tucker, E. B. (1989) Symplastic transport of carbxyfluorescin in staminal hairs of Setcreasea purpurea is diffusive and includes loss to the vacuole. Plant Physiol. 90, 1143-1147; Robards, A. W., Lucas, W. J. (1990) Plasmodesmata. Annu. Rev. Plant PhysioL Plant Mol. Biol. 41, 369-419]. In many species, however, the actual process involved in loading into the sieve element-companion cell complex may involve an apoplasmic step [van Bel, A. J. E. (1992) Pathway and mechanisms of phloem loading. In: Carbon partitioning within and between organisms (eds. Pollock, C. J., Farrar, J. F., Gordon, A. J.) pp. 53-70. BIOS Scientific Publishers, Ltd., Oxford]. Furthermore, it remains to be elucidated whether the loading process constitutes the rate-determining step, or major control site, in the export of recently fixed photosynthate.
Experimental control over plasmodesmal SEL has recently been achieved using expression of viral movement proteins (MPs) in transgenic plants. In transgenic tobacco expressing the MP of tobacco mosaic virus (TMV-MP), this movement protein becomes localized to mesophyll plasmodesmata [Atkins, D., Hull, R., Wells, B., Roberts, K., Moore, P., Beachy, R. N. (1991) The tobacco mosaic virus 30K movement protein in transgenic tobacco plants is localized to plasmodesmata. J. Gen. Virol. 72, 209-211; Ding et al. 1992, supra; Moore, P. J., Fenczik, C. A., Deom, C. M., Beachy, R. N. (1992) Developmental changes in plasmodesmata in transgenic tobacco expressing the movement protein of tobacco mosaic virus. Protoplasma 170, 115-127] where it causes a significant increase in plasmodesmal SEL from the control value of approx. 800 Da to greater than 9.4 kDa [Wolf et al. 1989, supra]. Photosynthesis and carbon allocation experiments performed on these transgenic tobacco plants revealed that the presence of the TMV-MP resulted in a change in carbon metabolism [Lucas et al. 1993, supra]. Although total chlorophyll, net photosynthesis and total leaf proteins were not significantly different between control and TMV-MP plant lines, it was found that, compared with control plants, fully expanded leaves expressing the TMV-MP had unexpectedly high levels of sugars and starch. The level of carbohydrates within these TMV-MP leaves appeared to increase more rapidly during the photoperiod, compared with control leaves, with the converse occurring during the dark period. In addition, there was a significant difference in biomass distribution between the various plant organs, resulting in lower root-to-shoot ratios in TMV-MP transgenic plants, although, under the growth conditions employed in these studies, the total biomass remained similar in both plant lines [Lucas et al. 1993, supra]. This complex influence of the TMV-MP on carbon metabolism was unexpected, since it was anticipated that increasing the plasmodesmal SEL would enhance symplasmic transport of sugars from the mesophyll to the site of phloem loading. If this were the case, sugar levels within the mesophyll tissue of TMV-MP plants should have been lower, not higher, than control values. Furthermore, if plasmodesmal SEL within the mesophyll did not constitute a rate-limiting step on the process of loading, carbon metabolism should have remained unaffected by the TMV-MP. Interpretation of these results was further complicated by the finding that although the TMV-MP was expressed in the vascular tissue, it did not cause an increase in the SEL of the plasmodesmata that interconnect the cells within the vein [Ding et al. 1992, supra].
It is an object of the present invention to provide compositions and methods which do not suffer from all of the drawbacks of the heretofore known compositions and methods.
In accordance with the present invention, there are provided methods and compositions for use in regulating plant metabolism and growth, wherein a plant regulatory composition (as hereinafter defined) is administered in a manner such that plasmodesmal transport of the composition in a predetermined manner is effected. Evidence is provided herein that plant encoded genes have the capacity to traffic via plasmodesmata to influence cell fate. In one example, TMV-MP is shown to interfere with an endogenous signal transduction pathway that involves macromolecular trafficking through plasmodesmata to regulate biomass partitioning between the source and various sink tissues. In another example, three homeotic proteins encoded by the maize homeobox gene Knotted-1and the MADS box genes deficiens and globosa of Antirrhinum [Sommer H., Nacken W., Beltran P., Huijser P., Pape H., Hansen R., Flor P., Saedler H., Schwartz-Sommer Z. (1991) Properties of deficiens, a homeotic gene involved in the control of flower morphogenesis in Antirrhinum majus. Development Supp 1, 169-175; Troebner W., Ramirez L., Motte P., Hue I., Huijser P., Loennig W.-E., Saedler H., Sommer H., Schwartz-Sommer Z. (1992) GLOBOSA: A homeotic gene which interacts with DEFICIENS in the control of Antirrhinum floral organogenesis. EMBO J. 11, 4693-4704] have now been shown to interact with plasmodesmata to mediate in their cell-to-cell transport. In yet another example, the first direct experimental proof that a plant mRNA-encoded protein can mediate in the plasmodesmal transport of itself and its own mRNA such that the mRNA can undergo extensive cell-to-cell movement is provided.
A further example illustrates that plant growth response to light intensity is altered by a viral movement protein. And in yet a different example, selective cell-to-cell movements of proteins through plasmodesmata are shown to potentiate cellular interactions between cells in adjacent cell layers, such as between layers of meristematic tissue and; between vascular tissue cells and cells in adjacent mesophyll and epidermal layers.